Listeria monocytogenes is a facultative intracellular bacterial pathogen that is an important agent of serious human food-borne infections. L. monocytogenes has served for decades as an outstanding model system for elucidating cellular and molecular interactions that take place during host infection. This proposal seeks to combine the power of bacterial genetics with the use of a genetically tractable model host system to isolate host mutants with altered resistance to L. monocytogenes infection. The fruit fly Drosophila melanogaster has been intensely studied as a model genetic system for decades, and it offers several advantages as a model host including the striking conservation of its innate immune recognition pathways with those of vertebrate animals. Studies have demonstrated the conservation of pathogenic mechanisms used by infectious agents within flies and vertebrates, and the feasibility of screening thousands of flies to isolate mutants with altered responses to infection provides a powerful means of identifying host factors that contribute to host survival. Recent work has demonstrated that D. melanogaster serves as a suitable host for L. monocytogenes infection, thus this proposal seeks to exploit the use of these genetically tractable organisms to functionally identify critical factors of both pathogen and host that contribute to the establishment of microbe infection. In Aim 1, experiments will examine the cellular course of L. monocytogenes infection within insect tissue culture cells, larvae, and adult flies. These studies will provide a foundation for the functional analysis of pathogen and host gene products identified for their potential roles in influencing the outcome of microbial infection. Aim 2 will functionally characterize L. monocytogenes mutants that are attenuated for virulence in flies. Bacterial gene products required for insect infection will be analyzed for potential roles in mammalian infection. Aim 3 will isolate and identify Drosophila mutants with altered susceptibility to Listeria infection. These studies will help identify the host factors that contribute to immune responses directed against intracellular pathogens. The ultimate goal of the experiments described will be the functional characterization of bacterial factors that support survival within the host, and the elucidation of host mechanisms that serve to counter bacterial survival strategies.